This revised proposal seeks to extend previously funded studies of the immunobiology of melanoma. It is hoped to continue to explore and understand the nature and significance of tumor-associated antigens that are immunogenic to the host. Through dissociation of Immune complexes by acidification and ultrafiltration (AD&U) of sera, it has been possible to augment autologous antibody activity against melanoma in the majority of systems studied. This provides a probe through which melanoma-associated antigens, not apparent to heteroantisera, may be identified and isolated. Previous work has identified a 66 kD melanoma-associated antigen present in serum and spent tissue culture media. Preliminary characterization through the utilization of an Isoelectric focusing column (IEF) and HPLC ion exchange chromatography (HP-IEC) have determined the conditions by which the antigen, present in spent media, may be preparatively purified. The antigen has been found to be unusually acidic (pl 2-3) and is sensitive to trypsin and neuraminidase digestion. Binding to three biotinylated lectins was noted as well. Preparative isolation of the antigen has been performed using anion exchange followed by lectin affinity chromatography. Following lectin affinity chromatography a single protein band at 66 kD was noted by SDS-PAGE and silver stain. Using this methodology, it is planned to obtain 1-2 mg of purified antigen. The amino acid sequence of the antigen will be determined by use of a gas phase sequenator available at Carnegie-Mellon University. Analysis of the carbohydrate composition will involve several approaches: 1) determination of the glycan- protein linkage; 2) evaluation of the effect of glycosylation inhibitors on antigen expression; 3) competitive binding with haptenic soluble sugars and their derivatives; 4) specific cleavage of terminal sugars. A monoclonal antibody directed against the intact antigen will be developed and will be used to better determine the distribution of the antigen immunohistologically. In addition, rabbits will be immunized with purified antigen and polyclonal sera directed toward the antigen obtained. Specificity of derived antibodies will be determined as has previously been performed with autologous antibody. The distribution of the antibodies will be examined immunohistologically by avidin-biotin staining against a number of normal and neoplastic lesion. Evaluation will include examination of normal and neoplastic tissues with an emphasis placed on skin. Dermatologic evaluation will include normal skin, melanocytic lesions (lentigo; junctional, compound and dermal nevi; dysplastic nevi; melanoma) as well as other benign and malignant skin conditions. Non-dermatologic evaluation will include examination of a range of normal and malignant tissue. Tissue specimens will be provided through the Departments of Dermatology, Medicine and Pathology. Finally, partial fragmentation of the purified antigen will be performed and the antigenic epitope detected by autologous antibody will be isolated. The fragment containing the antigenic epitope will be analyzed for its peptide and carbohydrate composition. Analysis of melanoma antigens that are immunogenic in the host may provide additional insight into the nature of the host-tumor interaction with implications for host defenses and immunotherapy.